1. Field of the Invention
The present invention relates to (1) novel 6-peptidylamino-1-naphthalenesulfonamides and (2) novel 6-amino-1-naphthalenesulfonamides. More particularly, the invention relates to novel 6-peptidylamino-1-naphthalenesulfonamides which are fluorogenic substrates for proteolytic enzymes, and specifically for the proteases involved in fibrinolysis and blood coagulation. These compounds are useful as substrates for determining proteolytic enzyme activity and as inhibitors of the enzymes. The invention also relates to 6-amino-1-naphthalenesulfonamide which are fluorogenic products of a reaction between a proteolytic enzyme and a 6-peptidylamino-1-naphthalensulfonamide substrate.
2. Description of the Related Art
Many of the proteases involved in blood coagulation and fibrinolysis can be described as trypsin-like in that they are serine proteases which preferentially hydrolyze peptide, ester, or amide bonds in which a basic amino acid provides the carbonyl group of the scissile bond. The in vivo specificity of these enzymes is a complex function of a variety of structural factors including binding domains in the protease for specific amino acid side chains located on both the amino and carboxyl side of the targeted lysine or arginine residue in the substrate protein. The idea that short peptide substrates can be designed to incorporate enough information to discriminate among these proteases relies on the concept that each active site is comprised of a unique series of side chain binding pockets.
Many fluorogenic and chromogenic amino acid substrates for proteolytic enzymes have been reported for assaying proteases. Lottenberg et al., (Methods Enzymol., 80, 341-361, 1981) report kinetic data for a large number of peptide derivative protease substrates. These substrates included p-nitroanilide (4-nitroanilide) derivatives, thiobenzyl esters, and nitrobenzyl esters of peptide residues having arginine or lysine at the carboxy terminus. Data were also presented for 7-amino-4-methylcoumarin, 2-naphthylamide (.beta.-naphthylamide), and 5-aminoisophthalic acid peptide derivatives. The derivatives, where P represents a peptide attached via the carbonyl of its carboxy terminus, may be represented as follows: ##STR2##
Bell et al., (Anal. Biochem., 61, 200-208, 1974) reported a fluorometric assay for plasmin, plasminogen, and streptokinase using .alpha.-N-methyl, .alpha.-N-tosyl-L-lysine ester of .beta.-naphthol.
Nieuwenhuizen et al., (Anal. Biochem., 83, 143-148, 1977) analyzed .beta.-naphthylamide derivatives of peptides having arginine at the carboxy terminus for their use as substrates of plasmin, urokinase, and plasminogen activator.
Morita et al., (J. Biochem., 82, 1495-1498, 1977) examined peptide-4-methylcoumarins containing arginine at the carboxy terminus as substrates for .alpha.-thrombin, factor Xa, and kallikreins, urokinase, and plasmin.
Cho et al., (Biochemistry, 23, 644-650, 1984) used a series of tripeptide 4-nitroanilide substrates in mapping studies of the S.sub.3 subsite of several serine proteases involved in blood coagulation. These substrates were of the type Z-AA-Gly-Arg-NA and Z-AA-PHE-ARG-NA where AA represents an amino acid and NA is 4-nitroanilide.
McRae et al., (Biochemistry, 20, 7196-7206, 1981) mapped the subsite specificities of certain proteases using amino acid, dipeptide, and longer peptide thioester substrates.
Chem. Abstracts, 109: 110461c [Japanese patent No. 63017870 A2 (Hidaka et al.)] discloses hexahydro-1-(naphthylsulfonyl)-1H-1,4-diazepines as cardiovascular agents. The disclosed compounds have the formula: ##STR3## where R.sub.1 and R.sub.2 represent H, halogen, lower alkyl, lower alkoxy, OH, NH.sub.2, lower alkylamino, lower acylamino, NO.sub.2, and cyano.
Chem. Abstracts. 62: 1615b and c [Polish patent application No. 48,252 (Wojtkiewicz and Jankowski, 1963)] discloses the synthesis of sulfonamide derivatives of 2-naphthylamine, including 2-aminonaphthalene-5-sulfonamide(i, R,R'.dbd.H), 2-aminonaphthalene-5-(N-methyl)sulfonamide(i, R.dbd.CH.sub.3, R'.dbd.H), and 2-aminonaphthalene-5-(N,N-dimethyl)sulfonamide(i, R,R'.dbd.CH.sub.3). ##STR4##
Chem. Abstracts, 51: 4722a [Swiss patent No. 312,090] discloses the use of 2-amino-5-naphthalenesulfonic acid methylamide in the preparation of cobalt containing azo dyes.
Lawson and Mann, (J. Biol. Chem., 266: 11317-11327, 1991), which is incorporated herein by reference, describes the investigation of the activation of human coagulation factor IX by human tissue factor.factor VIIa.PCPS.Ca.sup.2+ and factor Xa. PCPS.Ca.sup.2 + enzyme complexes. It is suggested that factors IX and X, when presented to the tissue factor.factor VIIa.PCPS.Ca.sup.2+ complex, are both rapidly activated and that factor Xa, which is generated in the initial stages of the extrinsic pathway, participates in the first proteolytic step in the activation of factor IX, the generation of factor IXa.
Lawson et al., (J. Biol. Chem. 267: 4834-4843, 1992), which is incorporated herein by reference, discusses the development of a fluorescent substrate (6-Mes-D-Leu-Gly-Arg)amino-1-(diethyl)napthalenesulfonamide) which can be used to directly measure the enzymatic activity of factor VIIa in the presence and absence of tissue factor and phospholipid.
Butenas et al., (Biochem. 31: 5399-5411, 1992) which is incorporated herein by reference, describes 6-amino-1-naphthalenesulfonamides and 6-peptidylamino-1-naphthalenesulfonamides useful in the detection of serine proteases involved in coagulation and fibrinolysis.
Butenas et al., (Chemistry, [Lithuanian Academy of Sciences] 182: 144-153, 1992) which is incorporated herein by reference describes the synthesis of 6-amino-1-naphthalenesulfonamides and suggests that these compounds may be used as detecting groups in peptide substrates for proteases.
A major weakness with many of the existing synthetic substrates is their low rate of enzymatic hydrolysis. Thus, large amounts of enzyme are normally required for the assay. While these prior art substrates provide increased sensitivity over substrates designed to exploit absorbance changes between substrate and product, they are plagued with other problems: overlap between the fluorescence spectral properties of the substrate and its hydrolysis products, lack of adequate solubility of substrate and fluorescent product in aqueous buffer, rapid rate of nonenzymatic substrate hydrolysis, and stability of the generated fluorescent product to both photo and chemical decomposition.
Thus, fluorogenic protease substrates are needed that have high specificity for the desired proteolytic enzyme, minimal overlap between the fluorescent spectral properties of the substrate and the hydrolysis products, reasonable solubility of the substrate and fluorescent products in aqueous buffer, low rates of non-enzymatic hydrolysis, and stability of the generated fluorescent product to both photo and chemical lysis.